Gene transcription by RNA polymerase II (RNAPII) is an essential process for the growth and development of all organisms. Transcription is highly coordinated and regulated at three main stages: initiation, elongation, and termination. While initiation is fairly well understood, many aspects of elongation remain enigmatic. During elongation, a multitude of factors dynamically interact with the C-terminal domain (CTD) of the RNAPII largest subunit, which acts as a platform to guide elongation processes. One factor that is widely found across many actively-transcribed genes is Spn1, which is conserved from yeast to humans, is essential for viability, and interacts with other members of a general elongation complex. Despite its conservation and essential nature, Spn1 remains poorly understood. Using the highly tractable model organism, Saccharomyces cerevisiae, this proposal contains two aims to functionally characterize Spn1 by analyzing its molecular interactions with other members of the general elongation complex. Specific Aim 1 examines the interaction of Spn1 with Spt6, another conserved and essential elongation factor that strongly associates with Spn1. These factors have shown codependency for recruitment to chromatin during transcription at certain genes studied; however, the dependency relationship of this recruitment is not understood and may differ across the genome, with different consequences for transcription. Therefore, the recruitment profiles of these two factors will be analyzed genome-wide in wild-type and in mutants impaired for the Spn1:Spt6 interaction. Second, since Spn1 and Spt6 together have been implicated in Set2-dependent histone H3 trimethylation-important for transcriptional integrity-additional experiments will determine if there is a correlation between a certain mode of Spn1/Spt6 recruitment and Set2 function. Aside from its interaction with Spt6, Spn1 also interacts with the conserved elongation factor Elf1- genetically and within a general elongation complex-suggesting an important functional relationship. Furthermore, these two factors are both phosphorylated by casein kinase (CK2), which is known to regulate the functions of other factors involved in transcription. Dissecting the link between Spn1, Elf1, and their phosphorylation will likely provide new insights into the regulation of Spn1 function. To achieve this, sites targeted for CK2-mediated phosphorylation on Spn1 and Elf1 will be mapped by combining mutational analysis with in vitro phosphorylation assays. Next, the sites identified will be validated in vivo, and a requirement for Elf1 in Spn1 phosphorylation will be assessed. Finally, the functional relevance of Spn1 phosphorylation will be analyzed by measuring changes in transcription by RNA-seq. The functional characterization of Spn1 will contribute to our overall understanding of how transcription elongation is differentially regulated from gene to gene. Given the conservation of these factors and transcriptional processes, what is learned from these studies will help uncover how perturbations in transcription can lead to disease states.